Understanding the inherent heterogeneity within living systems demands the development of new in vivo single molecule (SM) optical methods to follow protein dynamics without the veil of ensemble averaging. Noble metal nanoclusters exhibit exceedingly strong, size dependent emission throughout the visible and near IR spectrum, but at much smaller sizes (<l-nm) than comparable semiconductor quantum dots. The high polarizability of metal nanoclusters leads to extremely short, high efficiency radiative lifetimes (approximately 30-ps, and quantum yield of approximately 50%), and even enhances the Raman signal from the encapsulating scaffold to make it observable on the single molecule level. We will continue using poly(amidoamine) dendrimers (PAMAM) to solubilize and stabilize these highly emissive nanoclusters. Through dendrimer synthesis to a) incorporate specific Raman active labels in background-free spectral regions and b) modular dendrimer functionalization for incorporating generalized dendrimer encapsulated nanocluster ('nanodot') biochemical functionality, we will develop materials that uniquely enable in vivo single molecule imaging. The unique photophysics (extremely fast radiative lifetime, high quantum yield, and ability to produce Raman signals without a large nanoparticle) make these sub-nm nanoclusters as strongly absorbing as much larger (3-10 nm) semiconductor quantum dots, but, because they are not limited by the long quantum dot radiative lifetime of approximately 10 ns, the nanodot emission rates, and therefore brightness are at least two orders of magnitude higher. We will fully characterize the optical response of this new class of important nanomaterials as we employ them as biological labels. Their advantageous properties enable time and spectrally gated detection to obtain very high single molecule signals even in the presence of high autofluorescent backgrounds characteristic of living cells. We have assembled an outstanding team to chemically functionalize the PAMAM scaffold encapsulating and stabilizing the highly emissive Au and Ag nanoclusters and optically and biochemically characterize them in vitro and in vivo. Through three specific Aims, we will develop these robust ultrabright and ultrasmall nanodots into unparalled, specific, in vivo biological labels. In Aim I we will use targeted chemical synthesis to incorporate modularity in the PAMAM scaffold such that any functional group for modular attachment of biochemical targeting and recognition units can be incorporated. In Aim II we will synthesize and attach membrane transport functionalities to the nanodots and characterize their uptake and optical properties. These studies lead to Aim III in which multifunctionalized nanodots are made to specifically bind fusion proteins within the cytosol and we gate their transport into specific organelles. The single molecule imaging methods will be developed such that these extremely bright probes can be directly imaged by temporally and spectrally rejecting essentially all background from the more long-lived (ns) autofluorescent species. These combined methods should be capable of increasing current signal/noise ratios by more than three orders of magnitude over current nanoparticle or organic fluorophore based methods. This toolbox of modular, ultrabright, ultrasmall, and short-radiative lifetime nanodots will be generally applicable to a wide range of systems and will be made available to the community through this Exploratory Center.